This year we have continued to identify novel mAbs in several formats as Fabs, scFvs and eAds against cancer-related proteins. These mAbs were tested for their activity against cancer cells in vitro and used for development of novel approaches for multispecific targeting. We have also developed several libraries of eAds which can be used as a source of binders to various targets. The major accomplishments are summarized below. 1) We have previously proposed to use isolated CH2 domains as scaffolds for construction of libraries containing diverse binders, and identified and characterized a stabilized scaffold containing an additional disulfide bond. We have previously shown that an isolated human CH2 domain is relatively unstable to thermally induced unfolding but its stability can be improved by engineering an additional disulfide bond. We have hypothesized that the stability of this engineered antibody domain can be further increased by removing unstructured residues. To test our hypothesis we removed the seven N-terminal residues that are in a random coil as suggested by our analysis of the isolated CH2 crystal structure and NMR data. The resulting shortened engineered CH2 (m01s) was highly soluble, monomeric and remarkably stable with a melting temperature (Tm) of 82.6C which is about 10C and 30C higher than that of the original stabilized CH2 (m01) and CH2, respectively. m01s and m01 were more resistant to protease digestion than CH2. A newly identified anti-CH2 antibody which recognizes a conformational epitope bound to m01s significantly better (>10-fold higher affinity) than to CH2 and slightly better than to m01. m01s bound to a recombinant soluble human Fc neonatal receptor at pH 6.0 more strongly than CH2. These data suggest that shortening of the m01 N-terminus significantly increases stability without disrupting its conformation, and that our approach for increasing stability and decreasing size by removing unstructured regions may also apply to other proteins. 2) Novel yeast-displayed libraries of engineered CH2 domains were constructed and are being tested for selection of binders. Several binders against cancer-related proteins were identified and are being characterized. 3) We are also improving CH2-based scaffolds for binding to human neonatal Fc receptor (FcRn) with the specific aim to generate nAbs that can bind specifically to a cancer-related protein and simultaneously to the FcRn. The FcRn is responsible for transporting maternal IgGs to fetus/newborns and maintaining the homeostasis of IgGs in adults. FcRn resembles class I major histocompatibility complex in structure, and is composed of a transmembrane heavy chain and an invariant beta 2 microglobulin. Changes in the affinity of IgGs to FcRn lead to changes in the half-life of engineered IgGs and Fc fusion proteins. Longer half-life of therapeutic antibodies means lower dose and longer interval between administering. For some diagnostic agents including imaging or radio-labeled agents a shorter half life in circulation results in lower non-specific binding and decreased side effects. Therefore, studying the interaction of FcRn and therapeutic antibodies has direct clinical implications. A reliable method to prepare soluble and functional FcRn protein is essential for such studies. We developed a new method to express in mammalian cells soluble human FcRn (sFcRn) as a single-chain soluble fusion protein. The highly hydrophilic beta 2 microglobulin was joined with the hydrophobic heavy chain via a 15 amino acid linker. The single-chain fusion protein format not only improved the expression level of the heavy chain but also simplified the purification process. The sFcRn maintained its pH-dependent binding to IgG. This method typically yielded about 1mg/100ml culture without optimization, and is easy to scale up for production of large quantities. 4) We used our VH-based libraries for selection of eAds against cancer-related proteins, and the selected binders are being characterized. 5) We have extensively characterized our new mAb, m909, against the folate receptor beta (FRbeta). FRbeta is only detectable in placenta and limited to some hematopoietic cells of myeloid lineage in healthy people. Studies have indicated that FRbeta is over-expressed in activated macrophages in autoimmune diseases and some cancer cells. Functional recombinant FRbeta protein was produced in insect cells and used as antigen to isolate a mAb, m909, from a human naive Fab phage display library. Binding of Fab and IgG1 m909 to FRbeta was measured by ELISA, surface plasmon resonance, immune fluorescence staining, and flow cytometry. Antibody-dependent cell-mediated cytotoxicity (ADCC) was evaluated with FRbeta positive CHO cells as target cells and isolated peripheral blood monocytes as effector cells in an in vitro assay. Fab m909 bound with relatively high affinity (equilibrium dissociation constant 57 nM) to FRbeta. The IgG1 m909 showed much higher (femtomolar) avidity as measured by ELISA, and it bound to FRbeta positive cells in a dose-dependent manner, but not to parental FRbeta negative cells. m909 did not compete with folate for the binding to FRbeta on cells. m909 was not only able to select FRbeta positive, activated macrophages from synovial fluid cells of arthritis patients as efficiently as folate, but also able to mediate ADCC in FRbeta positive cells. Unlike folate-drug conjugates, m909 selectively binds to FRbeta, does not recognize FRalpha, and has at least one effector function. m909 alone has potential to eliminate FRbeta positive cells. Because m909 does not compete with folate for receptor binding, it can be used with folate-drug conjugates in a combination therapy. m909 can also be a valuable research reagent. 6) The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its functional receptors, DR4 and DR5, have been established as promising targets for cancer treatment. Therapeutics targeting TRAIL and its receptors are not only effective in killing many types of tumors, but they also synergize with traditional therapies and show efficacy against tumors that are otherwise resistant to conventional treatments. We identified and characterized two human monoclonal antibodies, m921 and m922, that are specific for human DR4. Both antibodies competed with TRAIL for binding to DR4, but only m921 recognized cell surface-associated DR4 and inhibited the growth of ST486 cells. This antibody may have potential for further development as candidate therapeutics and research tools. 7) We have continued to analyze high-throughput sequencing of large portions of the antibody repertoires of humans (the human antibodyome) and our libraries of binders. We believe that knowledge of the complete antibodyome will have implications for research, diagnosis, prevention and treatment of cancer. It can help for deeper understanding of the B cell (system) biology and diseases, help develop new diagnostic methods based on individual antibodyomes, help predict individual immune responses to immunization and therapeutics as well as provide information for the design of novel therapeutics. Most implications can not be predicted due to the very nature of an omic science.